This invention is in the field of biology. In particular, this invention relates to the cloning and expression of the C-terminal 42 kDa fragment of the Major Merozoite Surface Protein 1 (MSP142) or antigenic fragments thereof in a silkworm baculovirus expression system, the purification of the MSP142 and the production of a vaccine therefrom.
Malaria is caused by protozoal parasites of the genus Plasmodium. There are four species that infect man, P. falciparum P, vivax, P. malariae and P. ovale. Of these P. falciparum is largely responsible for acute and often fatal malaria, but there is significant morbidity associated with each malaria infection and a large proportion of the world""s population is at risk from the disease. It has been estimated that malaria is a public health problem in areas where 40% of the world""s population live and the disease has severe social and economic consequences for these communities. There has been a recent resurgence of the disease due to the abandonment or breakdown of control measures and to an increasing resistance of the vector to insecticides and falciparum malaria to chemotherapy.
Thus, there is an urgent need to develop a vaccine effective against malaria.
In order to meet these needs, the present invention is directed to the use of a baculovirus expression system to produce a recombinant C terminal 42 kDa fragment of the Major Merozoite Surface Protein (MSP142) for use in a malaria vaccine. The expression system of the invention is silkworms infected with recombinant nuclear polyhedrosis virus (NPV). The silkworms are preferably Bombyx mori silkworms (BmNPV). The malaria vaccine finds use in treating and preventing malaria including malaria resulting from the four species of the protozoal parasites of the genus Plasmodium that infect humans: P. falciparum P, vivax, P. malariae and P. ovale. 
The present invention is further directed to a method of producing a malaria vaccine, comprising: (a) expressing an immunogenic fragment of MSP 142 in a baculovirus expression system; (b) purifying the immunogenic fragment; and (c) formulating the immunogenic fragment in a malaria vaccine. In the method, the immunogenic fragment may include all or a portion of the MSP 142 protein. The MSP 142 protein may include a hexa-histidine tail.
In the method of purifying a malaria vaccine of the invention, the immunogenic fragment may be purified by chromatography or electrophoresis. The chromatography purification method may be ion exchange chromotogaphy, metal chelate affinity chromatography; molecular weight sieving, high pressure liquid chromatography, affinity chromatography or antibody affinity chromatography. The electrophoresis procedure may be agarose, acrylamide or isoelectric focusing electrophoresis.
In the method of producing a malaria vaccine of the invention the vaccine may include an adjuvant. The adjuvant may be selected from the group consisting of aluminum phosphate, aluminum hydroxide, saponin, Quil A, muramyl dipeptide, monophosphoryl lipid A muramyl tripeptide, cytokines, diphteriatoxoid, exotoxin A, granulocyte-macrophage colony stimulating factor and phospholipid conjugates. The adjuvant may further be selected from the group consisting of Adjumer(trademark); PCPP salt; polyphophazene; polyidi(carboxylatophenoxyl)phosphazene; Adju-Phos; Aluminum phosphate gel; xcex2-glucan; glucan; Gamma inulin/alum composite adjuvant; aluminum hydroxide gel; alum; N,N-dioctadecyl-N1, N1-bis(2-hydroexyethyl) propanediamine; N-(2-Deoxy-2-L-leucylamino-xcex2-D-glucopyranosyl)-N-octadecyldodecanoylamide; Calcitriol; 25-dihydroxyvitamin D3; 1,25-di(OH)2D3; 1,25-DHCC; 1xcex1,25-dihdroxycholecalciferol; 9,10-seco(5Z,7E)-5,7,10(19)-cholestatriene-1xcex1,3xcex2,25-triol; Block Copolymer P1205; Cytokine-containing Liposomes; Cytokine-containing Dehydration Rehydration Vesicles; Dimethyl dioctadecylammonium bromide; demethyl distearylammonium bromide; Dehydroepiandrosterone; 5-androsten-3xcex2-o1-17-one; dehydroisoandrosterone; androstenolone; prasterone; transdehydroandrosterone; Dimyristoyl phosphatidyl choline; sn-3-phosphatidyl choline-1,2-dimyristoyl; 1,2-dimyristoyl-sn-3-phosphatidyl choline; Dimyristoyl pposphatidylglcerol; sn-3-phosphatidyl glycerol-1,2-dimyristoyl, sodium salt; 1,2-dimyritoyl-sn-3-phosphatidyl glycerol; Deoxycholic Acid Sodium Salt; Gamma Inulin; Interleukin-1xcex2; IL-10; IL-1; human Interleukin 1xcex2 mature polypeptide Interferon-xcex3; Immunoliposomes Containing Antibodies to Costimulatory Molecules; ImmTher(trademark); N-acetylglucosaminyl-N-acetyhnuramyl-L-Ala-D-isoGlu-L-Ala-Glycerol dipalmitate; Imiquimod; 1-(2-methypropyl)-IH-imidazo[4,5-c]quinolin-4-amine; GMDP; N-acetylglucosaminyl-(xcex21-4)-N-acetylmuramyl-L-alanyl-D-isoglutamine; Gerbu Adjuvant; Interleukin-2; IL-2; T-cell growth factor; aldesleukin (des-alanyl-1, serine-125 human interleukin 2); Proleukin(copyright); Teceleukin(copyright); Interleukin-7; IL-7; Interleukin-12; IL-12; natural killer cell stimulatory factor (NKSF); cytotoxic lymphocyte maturation factor (CLMF); ISCOM(s)(trademark); Immune stimulating complexes; Iscoprep 7.0.3.(trademark); Liposomes; Liposomes (L) containing protein or Th-Cell and/or B-cell peptides, or microbes with or without co-entrapped interleukin-2, BisHOP or DOTMA; Loxoribine; 7-allyl-8-oxoguanosine; LT-OA or LT Oral Adjuvant; E. coli labile enteroxtoxin protoxin; MONTANIDE ISA 720; metabolizable oil adjuvant; MPL(trademark); 3-Q-dsacyl-4xe2x80x2-monophosphoryl lipid A; 3D-MLA; MF59; MTP-PE; N-acetyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1,2-dipalmitoyl-sn-glcero-3-(hydroxy-phosphoryloxy)) ethylamide, mono sodium salt; MTP-PE Liposomes; MTP-PE Antigen presenting liposomes; Murametide; Nac-Mur-L-Ala-D-Gln-OCH3; Murapalmitine; Nac-Mur-L-Thr-D-isoGln-sn-glycerol dipalmitoyl; D-Murapalmitine; Nac-Mur-D-Ala-D-isoGln-sn-glcerol dipalmitoyl; NAGO; Neuraminidase-galactose oxidase; Non-Ionic Surfactant Vesicles; NISV.; Pleuran; PLGA, PGA, and PLA; Homo-and co-polymers of lactic and glycolic acid; Lactide/glycolide polymers; poly-lactic-co-glycolide; Pluronic L121; Poloxamer 401; PMMA; Polymethyl methacrylate; PODDS(trademark); Proteinoid microspheres; Poly rA; Poly rU; Poly-adenylic acid-poly-uridylic acid complex; Polysorbate 80; Tween 80; Sorbitan mono-9-octadecenoate poly(oxy-1,2-ethanediyl) derivatives; Protein Cochleates; QS-21; Stimulon(trademark) QS-21 Adjuvant; Rehydragel HPA; High Protein Adsobency Aluminum Hydroxide Gel; alum; Rehydragel LV; low viscosity alluminum hydroxide gel; alum; S-28463; 4-Amino-otec, -dimethyl-2-ethoxmethyl-1H-imidazo[4,5-c]quinoline-1-ethanol; SAF-1; Scalvo peptide; IL-1xcex2 163-171 peptide; Span 85; Arlacel 85, sorbitan trioleate; Specol; Marcol 52 (mineral oil, paraffins, and cycloparaffins, chain length 13-22 C atoms) Span 85 (emulsifier, sorbitan trioleate) Tween 85 (emulsfier, polyoxyethylene-20-trioleate); Squalane; Spinacane; Robane(copyright); 2,6,10,15,19,23-hexamethyltetracosane; Squalene; Spinacene; Supraene; 2,6,10,15,19,23-hexamethyl-2,6,10,14,18,22 tetracosahexaene; Stearyl Tyrosine; Octadecyl tyrosine hydrochloride; Theramide(trademark); N-acetylglucosaminyl-N-acetylinuramyl-L-Ala-D-isoGlu-L-Ala-dipalmitoxy propylmadie (DTP-DPP); Threonyl-MDP; Termurtide(trademark); [thr1]-MDP ; N-acetyl muramyl-L-threonyl-D-isoglutamine; Ty Particles; and Ty-VLPs, (Virus Like Particles)
In the method of purifying a malaria vaccine of the invention the vaccine may include cholera toxin. The cholera toxin may be cholera toxin subunit A or cholera toxin subunit B.
The present invention is also directed to a method of immunizing a patient to malaria, comprising (a) preparing a malaria vaccine by expressing an immunogenic fragment of MSP142 in a baculovirus expression system; (b) purifying the immunogenic fragment; (c) formulating the immunogenic fragment in a malaria vaccine; and (d) administering the vaccine to said patient.
In the method of of immunizing a patient to malaria, the immunogenic fragment may be purified by chromatography or electrophoresis. The chromatography purification method may be ion exchange chromotogaphy, molecular weight sieving, high pressure liquid chromatography, affinity chromatography or antibody affinity chromatography. The electrophoresis procedure may be selected from agarose, acrylamide and isoelectric focusing electrophoresis.
In the method of immunizing a patient to malaria, the vaccine may include an adjuvant. In the method of immunizing a patient to malaria, the vaccine may include cholera toxin. The cholera toxin may be cholera toxin subunit A or cholera toxin subunit B.
The present invention is also directed to a malaria vaccine produced by the method of: (a) expressing an immunogenic fragment of MSP142 in a bacculovirus expression system; (b) purifying the immunogenic fragment; and (c) formulating the immunogenic fragment in a malaria vaccine.
The vaccine may further include an adjuvant. The vaccine may include cholera toxin. The cholera toxin may be cholera toxin subunit A or cholera toxin subunit B.
The present invention is further directed to a method of treating a patient with malaria, compromising: (a)expressing an immunogenic fragment of MSP142 in a baculovirus expression system; purifying the immunogenic fragment; (c) formulating the immunogenic fragment in a malaria vaccine; and (d) administering the malaria vaccine to the patient.
In the method of treating a patient with a malaria vaccine the immunogenic fragment may be purified by chromatography or electrophoresis. The chromatography purification method may be ion exchange chromotogaphy, molecular weight sieving, high pressure liquid chromatography, affinity chromatography and antibody affinity chromatography. The electrophoresis procedure may be selected from agarose, acrylamide and isoelectric focusing electrophoresis.
In the method of treating a patient with a malaria vaccine of the invention the vaccine may include an adjuvant. In the method of treating a patient with a malaria vaccine of the invention the vaccine may include cholera toxin. The cholera toxin may be cholera toxin subunit A or cholera toxin subunit B.
The invention is further directed to a method of purifying an immunogenic fragment of MSP142, comprising: expressing the immunogenic fragment in a bacculovirus expression system wherein the baculovirus expression system is Bombyx mori silkworms infected with nuclear polyhedrosis virus and purifying the immunogenic fragment by chromatography or electrophoresis.
In the method purifying an immunogenic fragment of MSP142, the chromatography purification method may be selected from ion exchange chromotogaphy, molecular weight sieving, high pressure liquid chromatography, affinity chromatography and antibody affinity chromotography. and the electrophoresis procedure may be agarose, acrylamide or isoelectric focusing electrophoresis.
The present ivention is further directed to an isolated and purified immunogenic fragment of MSP142 purified by the methods of the invention.
The present invention is further directed to a silkworm capable of expressing recombinant MSP 142. The silkworm may be Bombyx mori silkworm infected with recombinant nuclear polyhedrosis virus.